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1.
Chem Commun (Camb) ; 55(61): 8963-8966, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290488

RESUMO

We develop a simple method for sensitive detection of alkaline phosphatase (ALP) based on the ligase amplification reaction-catalyzed assembly of a single quantum dot (QD)-based nanosensor. This nanosensor requires only a single ligase enzyme to achieve ultrahigh sensitivity with a detection limit of 5.63 × 10-7 U mL-1, and can be applied for kinetic analysis, inhibitor screening, and ALP measurement in cell extracts.


Assuntos
Fosfatase Alcalina/análise , Técnicas Biossensoriais/métodos , DNA Ligases/química , Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Pontos Quânticos/química , Biotina/química , Carbocianinas/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluorescência , Células HEK293 , Humanos , Limite de Detecção , Células MCF-7 , Hibridização de Ácido Nucleico , Imagem Individual de Molécula , Estreptavidina/química
2.
Talanta ; 201: 450-454, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122448

RESUMO

A novel aggregation-induced emission (AIE) probe comprised of a hydrophilic protein kinase specific peptide and a hydrophobic tetraphenylethene (TPE) unit was synthesized through click reaction. The prepared TPE-peptide probe could be completely degraded by carboxypeptidase Y (CPY) to release hydrophobic TPE part, which aggregated in buffer solution and showed strong TPE emission. In the presence of casein kinase (CKII), the phosphorylation of peptide prevented the complete degradation by CPY producing the nonemissive probe. Thus, the developed probe can be used to detect CKII homogeneously and conveniently. This detection process can be finished within 1.5 h with high sensitivity (0.05 mU/µL) and good selectivity. The developed method can also be used to screen protein kinase inhibitor even in a complex biological system.


Assuntos
Técnicas Biossensoriais/métodos , Caseína Quinase II/análise , Ensaios Enzimáticos/métodos , Espectrometria de Fluorescência/métodos , Compostos de Benzilideno/síntese química , Compostos de Benzilideno/química , Caseína Quinase II/química , Catepsina A/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Células HeLa , Humanos , Oligopeptídeos/síntese química , Oligopeptídeos/química , Fosforilação , Inibidores de Proteínas Quinases/química , Proteólise , Triazóis/química
3.
Nat Commun ; 10(1): 2222, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110237

RESUMO

Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories. The high-resolution structure of the native Hordeum exo-hydrolase HvExoI isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl ß-D-glucoside and methyl 6-thio-ß-gentiobioside substrate analogues perfused in crystalline HvExoI bind across the catalytic site after they displace glucose, while methyl 2-thio-ß-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExoI reveal that upon productive binding of incoming substrates, the glucose product modifies its binding patterns and evokes the formation of a transient lateral cavity, which serves as a conduit for glucose departure to allow for the next catalytic round. This path enables substrate-product assisted processive catalysis through multiple hydrolytic events without HvExoI losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases.


Assuntos
Domínio Catalítico , Glucosidases/metabolismo , Modelos Moleculares , Proteínas de Plantas/metabolismo , Biocatálise , Cristalografia por Raios X , Ensaios Enzimáticos/métodos , Glucosidases/química , Glucosidases/isolamento & purificação , Glicosídeos/metabolismo , Hordeum/metabolismo , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Plântula/metabolismo , Especificidade por Substrato
4.
Biosens Bioelectron ; 138: 111308, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31103013

RESUMO

Poly(ADP-ribose) polymerase-1 (PARP-1), as an original tumor marker, has aroused wide attention in recent years. However, only a few researches have been done for PARP-1 activity detection because PARP-1 is lack of optical or electrochemical property. In this work, a label-free and high-sensitive photoelectrochemical (PEC) biosensor for PARP-1 activity detection based on poly[9,9-bis(6'-N,N,N-trimethylammonium)hexyl]fluorenylene phenylene (PFP) has been designed. To the best of our knowledge, it is the first time that PEC has been used to monitor PARP-1 activity. PARP-1 were activated under the function of activated dsDNA, as a result, branched polymers of ADP-ribose (PAR) with plentiful negative charge were formed in the presence of nicotinamide adenine dinucleotide (NAD+). Subsequently, positively charged PFP with good photoelectrochemical properties, were absorbed on PAR via electrostatic interaction. High photocurrent was produced under light induction, which was depended on the PARP-1 activity. The biosensor has a wide linear range from 0.01 to 2 U with a detection limit of 0.007 U. The strategy has been applied in breast and ovarian cancer cells to detection PARP-1 activity with approving results, which signifies that it is a promising tool for clinical diagnosis.


Assuntos
Biomarcadores Tumorais/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Linhagem Celular Tumoral , Técnicas Eletroquímicas , Eletrodos , Ensaios Enzimáticos/métodos , Humanos , Indóis/farmacologia , Limite de Detecção , NAD/farmacologia , Processos Fotoquímicos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Eletricidade Estática
5.
Talanta ; 199: 32-39, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952265

RESUMO

The majority of eukaryotic regulated protein turnover is performed by the proteasome, a multi-catalytic enzyme. Due to the fact that proteasome enzyme abnormal functioning was observed in different malignant cells, the proteasome is becoming a target for medical treatment. In order to evaluate the mechanisms of action of pharmaceutical compounds on proteasome enzyme inhibition, detecting and characterizing its activity is essential. An electrochemical assay that allows the monitoring of the chymotrypsin-like activity and inhibition of the 20S proteasome enzyme, based on the electrochemical detection of an electroactive compound released upon proteolysis of an adequate chymotrypsin-substrate is described. By employing differential pulse voltammetric measurement, the activity of the 20S proteasome enzyme was investigated for different incubation times of 20S with oligopeptide substrate as well as for different concentrations of substrate. Enzyme kinetic parameters were determined by voltammetry and the electrochemical assay compared with fluorescence spectroscopy. Electrochemical quartz crystal microbalance and atomic force microscopy were also used to investigate substrate interaction with the 20S proteasome and their adsorption at the electrode surface. Finally, the new electrochemical assay allowed to investigate the mechanisms of two different proteasome inhibitor drugs, bortezomib and oprozomib, underlying the applicability of the assay for understanding proteasome inhibitor action.


Assuntos
Antineoplásicos/farmacologia , Bortezomib/farmacologia , Técnicas Eletroquímicas , Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/farmacologia , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Antineoplásicos/química , Bortezomib/química , Inibidores Enzimáticos/química , Humanos , Microscopia de Força Atômica , Estrutura Molecular , Oligopeptídeos/química , Inibidores de Proteassoma/química , Técnicas de Microbalança de Cristal de Quartzo
6.
Plant Physiol Biochem ; 139: 325-332, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30947063

RESUMO

Cysteine is the first organic molecule generated during the assimilation of sulfate. As such, cysteine and its derivatives are always essential signal molecules and thus have important roles in the regulation of many plant processes. O-acetylserine (thiol) lyase (OASTL) catalyzes the last step of the biosynthesis of cysteine. At present, detailed and comprehensive work about these enzymes has only been reported from the plant Arabidopsis thaliana, though sporadic studies on OASTL have been conducted on other dicots, such as spinach and soybean. However, few reports on the functions of OASTLs in monocots have been found in the literature. Here in this study, we obtained four SiOASTL genes (SiOASTL7, SiOASTL8, SiOASTL9 and SiOASTL10) from foxtail millet and analyzed their potential functions. Phylogenetically, the four SiOASTL genes did not belong to any published subfamily of the OASTL genes; instead they constituted a new subfamily specific to the OASTL genes from monocots. In sequencing, we found that with the exception of the pseudogene SiOASTL8, proteins encoded by the other three genes exhibited high similarity with OASTL proteins from Arabidopsis, though the critical PLP-binding sites of both SiOASTL7 and SiOASTL10 were missing. The enzymatic activity assays demonstrated that SiOASTL9 has the ability to catalyze the biosynthesis of both cysteine and S-sulfocysteine, while SiOASTL7 and SiOASTL10 did not possess any previously reported catalyzing abilities. In addition, the gene expression pattern analysis showed that all four genes were widely expressed in various tissues of foxtail millet, and all had a preference in the leaves. Under abiotic stresses, the expression of these genes could be induced by salt and drought stress. Our finding that cadmium could only up-regulate the transcription of SlOASTL8 and SlOASTL9, further indicates the diversified responses of SiOASTLs to abiotic stresses.


Assuntos
Proteínas de Plantas/metabolismo , Setaria (Planta)/enzimologia , Setaria (Planta)/metabolismo , Ensaios Enzimáticos/métodos , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Setaria (Planta)/genética
7.
Nat Commun ; 10(1): 1804, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31000703

RESUMO

Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.


Assuntos
Caseína Quinase Iépsilon/metabolismo , Proteínas Desgrenhadas/metabolismo , Domínios PDZ/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Técnicas Biossensoriais , Caseína Quinase Iépsilon/genética , Proteínas Desgrenhadas/genética , Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência , Receptores Frizzled/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Microscopia de Fluorescência/métodos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oócitos , Fosforilação/fisiologia , Análise de Célula Única/métodos , Xenopus laevis
8.
Carbohydr Res ; 477: 20-25, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30933787

RESUMO

UDP-apiose, a donor substrate of apiosyltransferases, is labile because of its intramolecular self-cyclization ability, resulting in the formation of apiofuranosyl-1,2-cyclic phosphate. Therefore, stabilization of UDP-apiose is indispensable for its availability and identifying and characterizing the apiosyltransferases involved in the biosynthesis of apiosylated sugar chains and glycosides. Here, we established a method for stabilizing UDP-apiose using bulky cations as counter ions. Bulky cations such as triethylamine effectively suppressed the degradation of UDP-apiose in solution. The half-life of UDP-apiose was increased to 48.1 ±â€¯2.4 h at pH 6.0 and 25 °C using triethylamine as a counter cation. UDP-apiose coordinated with a counter cation enabled long-term storage under freezing conditions. UDP-apiose was utilized as a donor substrate for apigenin 7-O-ß-D-glucoside apiosyltransferase to produce the apiosylated glycoside apiin. This apiosyltransferase assay will be useful for identifying genes encoding apiosyltransferases.


Assuntos
Ensaios Enzimáticos/métodos , Pentosiltransferases/metabolismo , Açúcares de Uridina Difosfato/síntese química , Açúcares de Uridina Difosfato/metabolismo , Configuração de Carboidratos , Pentosiltransferases/genética , Açúcares de Uridina Difosfato/química
9.
Biosens Bioelectron ; 134: 117-122, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30981130

RESUMO

DNA methylation is catalyzed by DNA methyltransferase (MTase) and concerned with many biological processes including pathogenesis of various human diseases. The monitoring of MTase activity is thus of great significance in disease diagnosis and drug screening. Herein, we developed a facile way to synthesize biocompatible invertase enzyme modified metal-organic framework (Invertase/MOF) materials, and explored its application in constructing a dual-response Dam MTase sensor for the first time. By using them as signal probes, in which high density of metal sites could be electrochemically detected and invertase could hydrolyze sucrose into glucose for generation of glucometer signal output, dual-response for accurate detection of Dam MTase was realized. In the presence of Dam MTase, the methylation of hairpin probe 1 (HP1) occurred and thus caused the cleavage of HP1 assisted by a restriction endonuclease (DpnI) to produce the binding sequences. The binding sequences then hybridized with the electrode-assembled HP2 to expose their sticky termini which sequentially hybridized with the Invertase/MOFs-tethered capture probes. Finally, the electrodes were incubated with a sucrose solution, followed by the separate electrochemical and glucometer detection. The present assay brought good performance which could detect Dam MTase activity as low as 0.001 U mL-1 with wide linear range and good selectivity against other cytosine MTase (M.SssI MTase). Moreover, it also owns ability to be potentially applied for the inhibitors screening by utilization of 5-fluorouracil as an inhibitor model. The results imply that our proposed method provides a convenient platform for early cancer diagnosis and therapeutic applications.


Assuntos
Técnicas Biossensoriais/métodos , Ensaios Enzimáticos/métodos , Estruturas Metalorgânicas/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/análise , Automonitorização da Glicemia/métodos , Cobre/química , Enzimas Imobilizadas/química , Glucose/análise , Ouro/química , Humanos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , beta-Frutofuranosidase/química
10.
Analyst ; 144(10): 3364-3368, 2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-30982832

RESUMO

We herein devise a simple and label-free strategy to determine S1 nuclease activity by exploiting the target-induced inhibition of exponential strand displacement amplification (eSDA). In principle, a DNA probe that is designed to produce a large amount of duplexes through a process of eSDA, is degraded by the catalytic activity of S1 nuclease. This reaction blocks the initiation of eSDA, leading to the quite-reduced fluorescence of a double-stranded DNA specific fluorescent dye, SYBR Green I compared to the one in the absence of S1 nuclease. With this simple but novel approach, the S1 nuclease activity was selectively assayed with the high sensitivity. In addition, this system was successfully demonstrated to possess the capability to screen potential inhibitors against S1 nuclease.


Assuntos
Sondas de DNA/química , DNA/química , Endodesoxirribonucleases/análise , Ensaios Enzimáticos/métodos , DNA/genética , Sondas de DNA/genética , Endodesoxirribonucleases/química , Corantes Fluorescentes/química , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Compostos Orgânicos/química , Espectrometria de Fluorescência/métodos
11.
Mar Drugs ; 17(4)2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30974812

RESUMO

Chitosanase has attracted great attention due to its potential applications in medicine, agriculture, and nutraceuticals. In this study, P. mucilaginosus TKU032, a bacterial strain isolated from Taiwanese soil, exhibited the highest chitosanase activity (0.53 U/mL) on medium containing shrimp heads as the sole carbon and nitrogen (C/N) source. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, a chitosanase isolated from P. mucilaginosus TKU032 cultured on shrimp head medium was determined at approximately 59 kDa. The characterized chitosanase showed interesting properties with optimal temperature and thermal stability up to 70 °C. Three chitosan oligosaccharide (COS) fractions were isolated from hydrolyzed colloidal chitosan that was catalyzed by TKU032 chitosanase. Of these, fraction I showed the highest α-glucosidase inhibitor (aGI) activity (65.86% at 20 mg/mL); its inhibitory mechanism followed the mixed noncompetitive inhibition model. Fractions II and III exhibited strong 2,2-diphenyl1-picrylhydrazyl (DPPH) radical scavenging activity (79.00% at 12 mg/mL and 73.29% at 16 mg/mL, respectively). In summary, the COS fractions obtained by hydrolyzing colloidal chitosan with TKU032 chitosanase may have potential use in medical or nutraceutical fields due to their aGI and antioxidant activities.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Fatores Biológicos/biossíntese , Glicosídeo Hidrolases/isolamento & purificação , Oligossacarídeos/biossíntese , Paenibacillus/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Biocatálise , Fatores Biológicos/farmacologia , Quitosana/metabolismo , Crustáceos/química , Ensaios Enzimáticos/métodos , Depuradores de Radicais Livres/metabolismo , Depuradores de Radicais Livres/farmacologia , Proteínas Fúngicas/metabolismo , Inibidores de Glicosídeo Hidrolases/metabolismo , Inibidores de Glicosídeo Hidrolases/farmacologia , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Oligossacarídeos/farmacologia , Paenibacillus/isolamento & purificação , Estabilidade Proteica , Microbiologia do Solo , Especificidade por Substrato , alfa-Glucosidases/metabolismo
12.
Analyst ; 144(9): 3064-3071, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-30916676

RESUMO

DNA glycosylase (DG) plays a significant role in repairing DNA lesions, and the dysregulation of DG activity is associated with a variety of human pathologies. Thus, the detection of DG activity is essential for biomedical research and clinical diagnosis. Herein, we develop a facile fluorometric method based on the base excision repair (BER) mediated cascading triple-signal amplification for the sensitive detection of DG. The presence of human alkyladenine DNA glycosylase (hAAG) can initiate the cleavage of the substrate at the mismatched deoxyinosine site by endonuclease IV (Endo IV), resulting in the breaking of the DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate strand displacement amplification (SDA) to release primers. The released primers can further bind to a circular template to induce an exponential primer generation rolling circle amplification (PG-RCA) reaction, producing a large number of primers. The primers that resulted from the SDA and PG-RCA reaction can induce the subsequent recycling cleavage of signal probes, leading to the generation of a fluorescence signal. Taking advantage of the high amplification efficiency of triple-signal amplification and the low background signal resulting from single uracil repair-mediated inhibition of nonspecific amplification, this method exhibits a low detection limit of 0.026 U mL-1 and a large dynamic range of 4 orders of magnitude for hAAG. Moreover, this method has distinct advantages of simplicity and low cost, and it can further quantify the hAAG activity from HeLa cell extracts, holding great potential in clinical diagnosis and biomedical research.


Assuntos
DNA Glicosilases/sangue , Reparo do DNA , DNA/química , Ensaios Enzimáticos/métodos , Fluorometria/métodos , Sequência de Bases , DNA Polimerase Dirigida por DNA/química , Desoxirribonuclease IV (Fago T4-Induzido)/química , Fluorescência , Corantes Fluorescentes/química , Geobacillus stearothermophilus/enzimologia , Células HeLa , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Uracila-DNA Glicosidase/química
13.
Methods Mol Biol ; 1952: 127-142, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825172

RESUMO

Hyaluronidases are a group of enzymes responsible for the degradation of hyaluronan. They seem to be associated with a plethora of pathological conditions, as it has been showcased by numerous studies over the past years. The emerging role of hyaluronidases in various pathological states, especially cancer, is of a great interest. Thus, they are considered as important research targets.In this chapter the popular assay for hyaluronidase analysis in biological fluids is presented and discussed in detail. The assay is divided into two steps; the first is zymography that aims mainly to detect different hyaluronidase enzymes in a biological sample, and the second is the direct quantitative measurement of enzymatic activity by a microtiter plate assay. Both steps are characterized by high sensitivity, simplicity, and limited time consumption.


Assuntos
Eletroforese/métodos , Ensaios Enzimáticos/métodos , Hialuronoglucosaminidase/análise , Biotinilação , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/sangue , Hialuronoglucosaminidase/metabolismo , Hialuronoglucosaminidase/urina , Saliva/química , Coloração e Rotulagem/métodos
14.
Methods Mol Biol ; 1952: 193-199, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825175

RESUMO

To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localize them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrades a given substrate. Here we present an in situ gelatin zymography method that allows for a precise localization of active gelatin-degrading enzymes in a tissue section. In this method, dye-quenched gelatin is put on top of a tissue section. During an incubation period, active gelatinolytic enzymes will degrade the substrate and fluorescent signals are emitted from the locations of these enzymes.


Assuntos
Ensaios Enzimáticos/métodos , Gelatinases/metabolismo , Microscopia de Fluorescência/métodos , Microtomia/métodos , Animais , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Gelatina/análise , Gelatina/metabolismo , Gelatinases/análise , Humanos , Glândulas Mamárias Humanas/química , Glândulas Mamárias Humanas/enzimologia , Glândulas Mamárias Humanas/ultraestrutura , Camundongos , Especificidade por Substrato , Inclusão do Tecido/métodos , Fixação de Tecidos/métodos
15.
Methods Mol Biol ; 1952: 201-210, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825176

RESUMO

To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localize them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrades a given substrate. Here we present a protocol for real-time gelatin zymography that is very useful for the detection of gelatin-degrading proteases in tissue extracts. This method uses fluorescence-labeled gelatin and therefore we also present an easy, fast, and cheap method for labeling gelatin with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF).


Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Ensaios Enzimáticos/métodos , Gelatinases/metabolismo , Animais , Linhagem Celular , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Furanos/análise , Furanos/metabolismo , Gelatina/análise , Gelatina/metabolismo , Gelatinases/análise , Humanos , Rim/química , Rim/enzimologia , Fígado/química , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Pele/química , Pele/enzimologia
16.
Int J Mol Sci ; 20(7)2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30925705

RESUMO

Kallikrein 13 (KLK13) was first identified as an enzyme that is downregulated in a subset of breast tumors. This serine protease has since been implicated in a number of pathological processes including ovarian, lung and gastric cancers. Here we report the design, synthesis and deconvolution of libraries of internally quenched fluorogenic peptide substrates to determine the specificity of substrate binding subsites of KLK13 in prime and non-prime regions (according to the Schechter and Berger convention). The substrate with the consensus sequential motive ABZ-Val-Arg-Phe-Arg-ANB-NH2 demonstrated selectivity towards KLK13 and was successfully converted into an activity-based probe by the incorporation of a chloromethylketone warhead and biotin bait. The compounds described may serve as suitable tools to detect KLK13 activity in diverse biological samples, as exemplified by overexpression experiments and targeted labeling of KLK13 in cell lysates and saliva. In addition, we describe the development of selective activity-based probes targeting KLK13, to our knowledge the first tool to analyze the presence of the active enzyme in biological samples.


Assuntos
Ensaios Enzimáticos/métodos , Calicreínas/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Humanos , Cinética , Neoplasias/enzimologia , Biblioteca de Peptídeos , Peptídeos/química , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
17.
Chem Asian J ; 14(11): 1965-1969, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-30884161

RESUMO

Golgi endo-α-mannosidase (G-EM) catalyzes an alternative deglucosylation process for N-glycans and plays important roles in the post-endoplasmic reticulum (ER) quality control pathway. To understand the post-ER quality control mechanism, we synthesized a tetrasaccharide probe for the detection of the hydrolytic activity of G-EM based on a fluorescence quenching assay. The probe was labeled with an N-methylanthraniloyl group as a reporter dye at the non-reducing end and a 2,4-dinitrophenyl group as a quencher at the reducing end. This probe is hydrolyzed to disaccharide derivatives by G-EM, resulting in increased fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of disaccharide derivative present, allowing the G-EM activity to be evaluated easily and quantitatively.


Assuntos
Complexo de Golgi/enzimologia , alfa-Manosidase/metabolismo , Retículo Endoplasmático/metabolismo , Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Hidrólise , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Especificidade por Substrato
18.
Artigo em Inglês | MEDLINE | ID: mdl-30901734

RESUMO

Thiopurines are drugs widely used for the treatment of autoimmune conditions, inflammatory bowel disease or acute lymphoblastic leukemia. Determination of thiopurine methyltransferase activity (TPMT), a major determinant of thiopurines toxicity, has been suggested before implementing thiopurine treatment. An ultraperformance liquid chromatography (UPLC) method was developed and validated for the quantification of TPMT enzyme activity based on the conversion of 6-mercaptopurine (6-MP) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-L-methionine (SAM) as methyl donor in red blood cell lysates (RBC). This method was improved from a previous laborious high performance liquid chromatography (HPLC) method, using a lower volume of injection and with a shorter runtime. After incubation and protein precipitation 6-MMP was separated on a HSS-T3 (2.1 × 50 mm, 1.8 µm) column and monitored by UV detection (290 nm). A change on the organic solvent used to dissolve 6-MP resulted in a reduction of interference by endogenous or non-enzymatic methylated 6-MMP. A full validation of the 6-MMP assay was performed according to the FDA and EMA guidelines. The method was linear from 0.125 to 2 nmol/mL, with acceptable values of accuracy and precision. The method was applied in 106 patients treated with thiopurines whose TPMT activity was previously quantified by HPLC. Evaluation through Bland-Altman plot showed that TPMT activities were in agreement between both methods.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ensaios Enzimáticos/métodos , Eritrócitos/enzimologia , Metiltransferases/sangue , Metiltransferases/metabolismo , Monitoramento de Medicamentos , Humanos , Limite de Detecção , Modelos Lineares , Mercaptopurina/análogos & derivados , Mercaptopurina/metabolismo , Reprodutibilidade dos Testes , S-Adenosilmetionina/análise , S-Adenosilmetionina/metabolismo
19.
Methods Mol Biol ; 1943: 291-299, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838623

RESUMO

Cellular toxicity and/or cell death entail complex mechanisms that require multifaceted characterization. A detailed mechanistic assessment of cytotoxicity is essential for design and construction of more effective polycations for nucleic acid delivery. A single toxicity assay cannot stand alone in determining the type and extent of damage or cell death mechanism. In this chapter we describe a lactate dehydrogenase (LDH) assay for high-throughput screening that can be used as a starting point for further detailed cytotoxicity determination. LDH release is considered an early event in necrosis but a late event in apoptosis. An accurate temporal assessment of the toxic responses is crucial as late apoptosis may convert into necrosis as well as in situations where cell death is initiated without any visible cell morphological changes or responses in assays measuring late events, resulting in early ongoing toxicity being overlooked.


Assuntos
Ensaios Enzimáticos/métodos , L-Lactato Desidrogenase/metabolismo , Poliaminas/toxicidade , Testes de Toxicidade/métodos , Animais , Células Cultivadas , Humanos , Ácidos Nucleicos/genética , Transfecção/métodos
20.
Analyst ; 144(8): 2811-2819, 2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30882810

RESUMO

We present an integrated microfluidic device for quantifying intracellular materials at the single-cell level. This device combines a dual-well structure and a microfluidic control system. The dual-well structure includes capture wells (20 µm in diameter) for trapping a single cell and reaction wells (200 µm in diameter) for confining reagents. The control system enables a programmable procedure for single-cell analysis. This device achieves highly efficient trapping of single cells, overcoming the Poisson distribution, while affording sufficient biochemical reagents for each isolated reactor. We successfully utilized the presented device to monitor the catalytic interaction between intracellular alkaline phosphatase enzyme and a fluorogenic substrate and to quantify the intracellular glucose concentration of a single K562 cell based on an external standard method. The results demonstrate the feasibility and convenience of our dual-well array microfluidic device as a practical single-cell research tool.


Assuntos
Fosfatase Alcalina/análise , Corantes Fluorescentes/química , Glucose/análise , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/métodos , Oxazinas/química , Ensaios Enzimáticos/instrumentação , Ensaios Enzimáticos/métodos , Desenho de Equipamento , Fluorescência , Humanos , Células K562 , Limite de Detecção , Técnicas Analíticas Microfluídicas/instrumentação , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
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